Transgenic crop plants with improved stress tolerance

ABSTRACT

Disclosed herein are novel compositions of NF-YB proteins and recombinant DNA for expressing NF-YB proteins that are used to produce transgenic plants with enhanced yield and/or enhanced water deficit stress tolerance.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority under 35USC §119(e) of U.S.Provisional Application Ser. No. 60/816,086 filed Jun. 23, 2006, whichis herein incorporated by reference in its entirety.

INCORPORATION OF SEQUENCE LISTINGS

A Computer Readable Form of the Sequence Listing (on CD-ROM containingthe file named “54485B.ST25.txt” which is 70 KB as measured inMS-WINDOWS operating system and was created on Jun. 22, 2007) isincorporated herein by reference.

FIELD OF THE INVENTION

Disclosed herein are transgenic plant cells in seeds and plants withimproved stress tolerance and methods of making and using such cells,seeds and plants.

BACKGROUND OF THE INVENTION

Crop plant yield is reduced by any of a number of biotic or abioticstresses on such plants. Growers have used a variety of strategies tominimize adverse effects from stress. For instance, stress from weedscan be reduced by application of herbicides, stress from insects can bereduced by application of pesticides, stress from water deficit can bereduced by irrigation, stress from cold can be reduced by delayingplanting time, and stress from nutrient deficiency can be reduced bytreating with fertilizer.

The yield from a plant is influenced by environmental factors includingwater availability, exposure to cold or heat, availability of nutrientssuch as phosphorus and nitrogen, plant density, and the like. A plant'sresponse to such environmental stress can be influenced by internalgenetic mechanisms. An object of plant genetic engineering is to producenovel plants with agronomically, horticulturally, or economicallyimportant traits including increased tolerance to any of a variety ofenvironmental stresses.

Considering the complexity of water use in land plants, especiallyduring conditions that produce water deficit, relatively few genesspecifically associated with this aspect of physiology have beenidentified. The use of recombinant DNA expressing certain Hap3 CAAT boxDNA binding transcription factors for improving water deficit toleranceis disclosed in US 2005/0022266 A1. Hap3 transcription factors are alsoknown as NF-YB proteins which form complexes with NF-YA and NF-YCproteins in plants. These proteins are collectively referred to as NFYproteins. The amino acid sequence of a corn NF-YB protein is SEQ IDNO:28.

SUMMARY OF THE INVENTION

This invention provides novel plant chromosomal DNA comprising arecombinant polynucleotide that provides for expression of an NF-Yprotein that provides protection against water deficit stressconditions. Such plant chromosomal DNA is flanked by native plant DNA.To accommodate the vagaries of nature, embodiments of the plantchromosomal DNA can provide plants with improved yield as compared tocontrol crop plants when the plants are grown in water deficit stressconditions, as well as comparable or improved yield as compared tocontrol crop plants when grown in water sufficient conditions. Thisinvention also provides transgenic plant cells, plants, seeds and cropshaving the novel plant chromosomal DNA of this invention.

A characteristic of the plant chromosomal DNA segments used in thisinvention is the presence of a recombinant polynucleotide that encodesfor low expression, e.g. constitutive expression at a level close tobackground expression of a native NF-Y protein, i.e. where NF-Y proteinis produced in leaf cells at a level up to 40 picograms per microgram oftotal protein in plant leaf tissue cells or less, e.g. up to about 30 or20 picograms per microgram of total protein in plant leaf tissue cells.In other embodiments the NF-Y protein expressed from the recombinantpolynucleotide is in the range of 0.1 to 11 picograms per microgram oftotal protein in plant leaf tissue cells.

In some embodiments of the invention the recombinant polynucleotideprovides for expression of at least one NF-Y protein and a markerprotein. In another embodiment the recombinant polynucleotide providesfor expression of a single NF-Y protein.

In some embodiments the NF-Y protein expressed by the recombinantpolynucleotide is a native NF-YA, NF-YB or NF-YC protein. In otherembodiments the NF-Y protein expressed by the recombinant polynucleotideis an exogenous NF-YA, NF-YB or NF-YC protein, e.g. an NF-YB proteinfrom Arabidopsis thaliana. In yet, other embodiments the NF-Y proteinexpressed by the recombinant polynucleotide is a variant of a nativeNF-YA, NF-YB or NF-YC, e.g. in corn plant chromosomal DNA therecombinant polynucleotide expresses an variant NF-YB protein comprisingcontiguous amino acids from native corn DNA sequence.

The plant chromosomal DNA segments of this invention are provided byemploying any number of low level constitutive promoters for expressionof the NF-Y protein. Such promoters are readily identified and isolatedby those skilled in the art and can include a promoter selected from thegroup consisting of a rice alpha tubulin promoter, a rice actinpromoter, a PPDK mesophyll tissue-enhanced promoter, and a rubiscoactivase bundle sheath tissue-enhanced promoter.

Such plant chromosomal DNA of this invention is useful in transgenicplant cells of plants that are desired to exhibit water deficit stresstolerance. Such plant chromosomal DNA is useful in providing atransgenic crop of water deficit stress tolerant plants. e.g. where theharvested yield of said crop is enhanced over the yield of a crop ofcontrol plants not having said plant chromosomal DNA segment. Because ofthe unpredictability of rainfall and/or availability of irrigationwater, plants of this invention may be grown in a wide range of watersufficiency conditions, ranging from water sufficient conditions overthe lifetime of the plant to various levels of water deficit stressconditions over the lifetime of the plant. To accommodate such variationin water sufficiency, certain embodiments of this invention providetransgenic crops that exhibit increased harvested yield as compared tocontrol crop plants when the plants are grown in water deficit stressconditions, as well as comparable or increased yield as compared tocontrol crop plants when grown in water sufficient conditions. Suchcrops in include water deficit-tolerant plants of corn, cotton, soybean,sugarcane, switchgrass, rice, wheat, alfalfa, or canola plants. In oneaspect of the invention the plant chromosomal DNA is in a transgeniccorn plant and comprises recombinant polynucleotides for expressing anNF-YB protein which is a native corn protein or a variant thereof.

Another aspect of this invention provides transgenic pollen grainscomprising a haptoid derivative of a plant cell containing a plantchromosomal DNA segment of this invention. Another aspect of thisinvention is anti-counterfeit milled seed having, as an indication oforigin, a plant cell with said chromosomal DNA segment of thisinvention.

Still other aspects of this invention provide methods of improving waterdeficit stress tolerance and yield in a crop plant line comprisingproviding in the genome of a crop plant line a plant chromosomal DNAsegment of this invention.

Another method of this invention provides for the manufacture ofnon-natural, transgenic seed or propagules that can be used to produce acrop of transgenic plants with enhanced water deficit toleranceresulting from expression of an NF-YB protein from a plant chromosomalDNA segment of this invention. Such a method comprises screening apopulation of plants having such plant chromosomal DNA segment andcontrol plants for said enhanced yield when grown under water-deficitstressed and enhanced or comparable yield when grown under watersufficient conditions, selecting from said population one or more plantsthat exhibit enhanced yield as compared to the yield for control plantsunder water-deficit stressed or enhanced or comparable yield as comparedto the yield for control plants when grown under water sufficientconditions, verifying that said plant chromosomal DNA segment is stablyintegrated in said selected plants, analyzing leaf tissue of a selectedplant to determine the production of transgenic NF-YB protein at a levelup to 40 picograms of NF-YB protein per microgram of total protein insaid leaf tissue, and collecting seed or a regenerative propagule from aselected plant.

Another method provides for the production of inbred corn seedcomprising acquiring hybrid corn seed from a herbicide tolerant cornplant which also has stably-integrated, chromosomal DNA segment of thisinvention, introgressing the chromosomal DNA segment from said acquiredhybrid corn seed into a second corn line by allowing pollen grainscomprising a haploid derivative with said chromosomal DNA segment topollinate said second corn line to produce crossed seeds, producing apopulation of plants from crossed seeds (where a fraction of the seedsproduced from said pollination is homozygous for the chromosomal DNAsegment, a fraction is hemizygous, and a fraction does not have thechromosomal DNA segment), selecting corn plants which are homozygous andhemizygous for said chromosomal DNA segment by treating with anherbicide, collecting seed from herbicide-treated-surviving corn plantsand planting said seed to produce further progeny corn plants, andbackcrossing plants grown from said progeny seeds with said second cornline to produce an inbred corn line. The method can be further employedby crossing the inbred corn line with a third corn line to producehybrid seed.

Yet another aspect of this invention provides a method of growing acorn, cotton, soybean, sugarcane, switchgrass, rice, wheat, alfalfa, orcanola crop without irrigation water comprising planting seed havingplant cells with a plant chromosomal DNA segment of this invention,where the seeds are produced from plants that are selected for enhancedwater deficit stress tolerance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plasmid map for plant transformation vector pMON82754.

FIG. 2 is a plasmid map for plant transformation vector pMON63796.

FIG. 3 illustrates the yield performance of plants under water deficitstress conditions where different promoters are used in recombinantpolynucleotides for expressing an NF-Y protein.

FIG. 4 illustrates the yield performance of plants under watersufficient conditions where different promoters are used in recombinantpolynucleotides for expressing an NF-Y protein.

DETAILED DESCRIPTION OF THE INVENTION

Unless specifically defined, all technical and scientific terms usedherein have the same meaning as commonly understood by persons ofordinary skill in the art. The procedures for preparing and screeningtransgenic plants described below are well known and commonly employedby persons of ordinary skill in the art.

As used herein “water deficit” means a period when water available to aplant is not replenished at the rate at which it is consumed by theplant. A long period of water deficit is colloquially called drought.Lack of rain or irrigation may not produce immediate water stress ifthere is an available reservoir of ground water for the growth rate ofplants. Plants grown in soil with ample groundwater can survive dayswithout rain or irrigation without adverse affects on yield. Plantsgrown in dry soil are likely to suffer adverse affects with minimalperiods of water deficit. Severe water deficit stress can cause wilt andplant death; moderate drought can cause reduced yield, stunted growth orretarded development. Plants can recover from some periods of waterdeficit stress without significantly affecting yield. However, waterdeficit stress at the time of pollination can have an irreversibleeffect in lowering yield. Thus, a useful period in the life cycle ofcorn for observing water deficit stress tolerance is the late vegetativestage of growth before tasseling. Water deficit stress tolerance isdetermined by comparison to control plants. For instance, plants of thisinvention can survive water deficit stress with a higher yield thancontrol plants. In the laboratory and in field trials drought can besimulated by giving plants of this invention and control plants lesswater than is given to sufficiently-watered control plants and measuringdifferences in traits.

A suitable control plant may be a non-transgenic plant of the parentalline used to generate a transgenic plant herein. A control plant may insome cases be a transgenic plant line that includes an empty vector ormarker gene, but does not contain the recombinant polynucleotide of thepresent invention that is expressed in the transgenic plant beingevaluated. A control plant in other cases is a transgenic plantexpressing the gene with a constitutive promoter. In general, a controlplant is a plant of the same line or variety as the transgenic plantbeing tested, lacking the specific trait-conferring, recombinant DNAthat characterizes the transgenic plant. Such a progenitor plant thatlacks that specific trait-conferring recombinant DNA can be a natural,wild-type plant, an elite, non-transgenic plant, or a transgenic plantwithout the specific trait-conferring, recombinant DNA thatcharacterizes the transgenic plant. The progenitor plant lacking thespecific, trait-conferring recombinant DNA can be a sibling of atransgenic plant having the specific, trait-conferring recombinant DNA.Such a progenitor sibling plant may include other recombinant DNA.

As used herein “yield” of a crop plant means the production of a crop,e.g., shelled corn kernels or soybean or cotton fiber, per unit ofproduction area, e.g., in bushels per acre or metric tons per hectare,often reported on a moisture adjusted basis, e.g., corn is typicallyreported at 15.5% moisture. Moreover a bushel of corn is defined by lawin the State of Iowa as 56 pounds by weight, a useful conversion factorfor corn yield is: 100 bushels per acre is equivalent to 6.272 metrictons per hectare. Other measurements for yield are in common practice.

A transgenic “plant cell” means a plant cell that is transformed withstably-integrated, non-natural, recombinant polynucleotides, e.g. byAgrobacterium-mediated transformation or by bombardment usingmicroparticles coated with recombinant polynucleotides. A plant cell ofthis invention can be an originally-transformed plant cell that existsas a microorganism or as a progeny plant cell that is regenerated intodifferentiated tissue, e.g. into a transgenic plant withstably-integrated, non-natural recombinant polynucleotides in itschromosomal DNA, or seed or pollen derived from a progeny transgenicplant.

A “transgenic” plant or seed means one whose genome has been altered bythe stable incorporation of recombinant polynucleotides in itschromosomal DNA, e.g. by transformation, by regeneration from atransformed plant from seed or propagule or by breeding with atransformed plant. Thus, transgenic plants include progeny plants of anoriginal plant derived from a transformation process including progenyof breeding transgenic plants with wild type plants or other transgenicplants. The enhancement of a desired trait can be measured by comparingthe trait property in a transgenic plant which has recombinant DNAconferring the trait to the trait level in a progenitor plant. Althoughmany varieties of plants can be advantageously transformed withrecombinant DNA for expressing an NF-YB protein to provide water stresstolerance and/or enhanced yield, especially useful transgenic plantswith water stress tolerance include corn (maize), soybean, cotton,canola (rape), wheat, rice, alfalfa, sorghum, grasses such asswitchgrass, vegetables and fruits.

“Expressing a protein” means the function of a cell to transcriberecombinant DNA to mRNA and translate the mRNA to a protein. Forexpression the recombinant DNA usually includes regulatory elementsincluding 5′ regulatory elements such as promoters, enhancers, andintrons; other elements can include polyadenylation sites, transitpeptide DNA, markers and other elements commonly used by those skilledin the art. Promoters can be modulated by proteins such as transcriptionfactors and by intron or enhancer elements linked to the promoter.Promoters in recombinant polynucleotides can also be modulated by nearbypromoters. For example, the activity of a low constitutive promoter asused in this invention can be significantly increased to undesirablyhigh levels by the activity of a second highly expressing promoter inthe recombinant polynucleotide. For instance, there is disclosed in US2005/0022266 A1 a recombinant polynucleotide construct a transcriptionunit comprising a low level-expressing rice actin promoter operablylinked to DNA coding for an NF-YB protein followed by a transcriptionunit comprising a CaMV 35S promoter operably linked to DNA coding forthe nptII marker. Although the plants having such recombinantpolynucleotides in the chromosomal DNA exhibited water deficit stresstolerance, the CaMV 35S promoter is able to enhance the otherwise lowexpression of the nearby rice actin promoter to such high levels as tocause a reduction in yield in plants grown under water sufficientconditions. An aspect of this invention involves the use of recombinantpolynucleotides having promoters for expressing NF-Y proteins at lowlevels to avoid reduction in yield when plants are grown under watersufficient conditions.

“Recombinant polynucleotide” means a DNA construct that is made bycombination of two otherwise separated segments of DNA, e.g., bychemical synthesis or by the manipulation of isolated segments ofnucleic acids by genetic engineering techniques. Recombinant DNA caninclude exogenous DNA or simply a manipulated native DNA. RecombinantDNA for expressing a protein in a plant is typically provided as anexpression cassette which has a promoter that is active in plant cellsoperably linked to DNA encoding a protein, e.g. an NF-YB protein, linkedto a 3′ DNA element for providing a polyadenylation site and signal.Useful recombinant DNA also includes expression cassettes for expressingone or more proteins conferring herbicide tolerance and/or insectresistance. With reference to the sequence listing the DNA of variouspromoters are identified in Table 1 and the DNA encoding variousembodiments of NF-YB proteins are identified in Table 2. Certain genesencoding native NF-YB subunits are identified using “Gnnnn”nomenclature, e.g. the Arabidopsis thaliana G481 gene. A set of usefulpromoters is disclosed in Table 1 with reference to DNA in the sequencelisting for the promoter element including enhancer, leader and intronelements used in various illustrative embodiments. These and numerousother promoters that function in plant cells are known to those skilledin the art and available for use in alternative embodiments of thisinvention to provide for expression of NF-Y proteins in transgenic plantcells. TABLE 1 Promoter Expression (species of origin) SEQ IDUbiquitous - high in leaf but lower in reproductive 1 and 2 tissue(CaMV35S-enhanced) Ubiquitous - high in leaf but lower in reproductive 3tissue (CaMV35S) Epidermis, stomatal guard cells enhanced (rice) 4 Silkenhanced (corn) 5 Silk enhanced (sorghum) 6 Constitutive - low level(corn) 7 Bundle sheath enhanced (corn RUA) 8 Drought inducible (corn) 9Mesophyll enhanced (corn) 10 Drought inducible (corn) 11 Root enhanced(pea) 12 Ubiquitous (rice alpha tubulin) 13 Ubiquitous (rice actin1) 14Ubiquitous (rice actin1) 15 Root enhanced (corn NAS2) 16 Embryo (barley)17 Ubiquitous (Arabidopsis) 18 Drought inducible (Arabidopsis) 19 Greentissue enhanced (Arabidopsis) 20 Drought inducible (Arabidopsis) 21Drought inducible (Arabidopsis) 22 Drought inducible (Arabidopsis) 23Green tissue enhanced (Arabidopsis) 24 Root enhanced (Arabidopsis) 25Vascular tissue enhanced (Arabidopsis) 26 Chimeric bundlesheath/mesophyll enhanced (corn) 27 Ubiquitous (Arabidopsis EF-1 alpha)60 Seed (Glycine max) 61

TABLE 2 SEQ Arbitrary DNA Name ID Protein Features Corn NF-YB2-S83A 29In SEQ ID NO: 28 Serine at position 83 is changed to Alanine CornNF-YB2-123C 30 Methionine and amino acids 29-134 of SEQ ID NO: 28representing domains 1, 2, 3 and C of the protein Corn NF-YB2-23C 31Methionine and amino acids 68-134 of SEQ ID NO: 28 representing domains2, 3 and C of the protein Corn NF-YB2-C73:89S 32 In SEQ ID NO: 28Cysteines at positions 73 and 89 are changed to Serine CornNF-YB2-C73R:C89S 33 In SEQ ID NO: 28 Cysteine at position 73 changed toArginine, and Cysteine at position 89 is changed to Serine Corn NF-YB2-34 In SEQ ID NO: 28 Cysteine at position 73 is C73S:C89S:L102R changedto Serine, Cysteine at position 89 is changed to Serine, and Leucine at102 is changed to Arginine Corn NF-YB2-E76R:S83R 35 In SEQ ID NO: 28Glutamate at position 76 is changed to Arginine and Serine at position83 is changed to Arginine Corn NF-YB2-I115A 36 In SEQ ID NO: 28Isoleucine at position 115 is changed to Alanine Corn NF-YB2- 37 In SEQID NO: 28 Isoleucine at position 49 is I49R:C73R:C89S:L102R changed toArginine, Cysteine at position 73 is changed to Arginine, Cysteine atposition 89 is changed to Serine, and Leucine at position 102 is changedto Arginine Corn NF-YB2- 38 In SEQ ID NO: 28 Isoleucine at position 49is I49R:C73S:C89S changed to Arginine, Cysteine at position 73 ischanged to Serine, and Cysteine at position 89 is changed to Serine CornNF-YB2-L102A 39 In SEQ ID NO: 28 Leucine at position 102 is changed toAlanine Corn NF-YB2-L103A 40 In SEQ ID NO: 28 Leucine at position 103 ischanged to Alanine Corn NF-YB2-L109A 41 In SEQ ID NO: 28 Leucine at 109is changed to Alanine Corn NF-YB2-L118A 42 In SEQ ID NO: 28 Leucine at118 is changed to Alanine Corn NF-YB2-L122A 43 In SEQ ID NO: 28 Leucineat 122 is changed to Alanine Corn NF-YB2 (PHE0000004) 44 NF-YB proteinof SEQ ID NO: 28 Corn NF-YB2a (PHE0008666) 45 Amino acids TPIANGK at55-61 of SEQ ID NO: 28 are deleted Corn.NFB2-1:4:9 46 Corn NF-YB2protein (PHE0008660) soybean G482-like 3 47 Soybean NF-YB protein(PHE0001202) Soybean NF-YB (G481 like) 48 Soybean NF-YB protein (G3472)Gm.G481-1:1:1 (PHE0010412) 49 Soybean NF-YB protein soy G481-like 3(PHE0003740) 50 Soybean NF-YB protein soybean G482-like 1 51 SoybeanNF-YB protein (PHE00001201) Soy G1820 like (PHE0003227) 52 Soybean NF-YBprotein Arabidopsis G481 53 Arabidopsis NF-YB protein (PHE0000002)Arabidopsis G1364 54 Arabidopsis NF-YB protein (PHE0003728) ArabidopsisG485 55 Arabidopsis NF-YB protein (PHE0010350) GhG481-1:1:1 (PHE0010352)56 Cotton NF-YB protein Gh.NFB2-1:1:1 (PHE0010354) 57 Cotton NF-YBprotein CR-Gm.G481-6-1:1:1 58 Soybean NF-YB protein (PHE0003701)Os.NFYB2 (PHE0004246) 59 Rice NF-YB protein

Recombinant DNA constructs generally include a 3′ element that typicallycontains a polyadenylation signal and site. Well-known 3′ elementsinclude those from Agrobacterium tumefaciens genes such as nos 3′, tml3′, tmr 3′, tms 3′, ocs 3′, tr7 3′, e.g., disclosed in U.S. Pat. No.6,090,627. 3′ elements from plant genes such as wheat (Triticumaestivum) heat shock protein 17 (Hsp17 3′), a wheat ubiquitin gene, awheat fructose-1,6-biphosphatase gene, a rice glutelin gene, a ricelactate dehydrogenase gene and a rice beta-tubulin gene are disclosed inU.S. published patent application 2002/0192813 A1.

Constructs and vectors may also include a transit peptide for targetingof a gene target to a plant organelle, particularly to a chloroplast,leucoplast or other plastid organelle. The use of chloroplast transitpeptides is disclosed in U.S. Pat. Nos. 5,188,642 and 5,728,925.

The plants of this invention can be further enhanced with stackedtraits, e.g., a crop having an enhanced agronomic trait resulting fromexpression of DNA disclosed herein, in combination with herbicide and/orpest resistance traits. For example, genes of the current invention canbe stacked with other traits of agronomic interest, such as a traitproviding herbicide resistance, or insect resistance, such as using agene from Bacillus thuringiensis to provide resistance againstlepidopteran, coleopteran, homopteran, hemiopteran, and other insects.Herbicides for which resistance is useful in a plant include glyphosateherbicides, dicamba herbicides, phosphinothricin herbicides, oxynilherbicides, imidazolinone herbicides, dinitroaniline herbicides,pyridine herbicides, sulfonylurea herbicides, bialaphos herbicides,sulfonamide herbicides and glufosinate herbicides. Persons of ordinaryskill in the art are enabled in providing stacked traits by reference toU.S. 2003/0106096A1 and 2002/0112260A1 and U.S. Pat. Nos. 5,034,322;5,776,760; 6,107,549 and 6,376,754 and to insect/nematode/virusresistance by reference to U.S. Pat. Nos. 5,250,515; 5,880,275;6,506,599; 5,986,175 and U.S. 2003/0150017 A1.

Plant Cell Transformation Methods

Numerous methods for transforming plant cells with recombinant DNA areknown in the art and may be used in the present invention. Two commonlyused methods for plant transformation are Agrobacterium-mediatedtransformation and microprojectile bombardment. Microprojectilebombardment methods are illustrated in U.S. Pat. No. 5,015,580(soybean); U.S. Pat. No. 5,550,318 (corn); U.S. Pat. No. 5,538,880(corn); U.S. Pat. No. 5,914,451 (soybean); U.S. Pat. No. 6,160,208(corn); U.S. Pat. No. 6,399,861 (corn) and U.S. Pat. No. 6,153,812(wheat) and Agrobacterium-mediated transformation is described in U.S.Pat. No. 5,159,135 (cotton); U.S. Pat. No. 5,824,877 (soybean); U.S.Pat. No. 5,591,616 (corn); and U.S. Pat. No. 6,384,301 (soybean), all ofwhich are incorporated herein by reference. For Agrobacteriumtumefaciens based plant transformation system, additional elementspresent on transformation constructs will include T-DNA left and rightborder sequences to facilitate incorporation of the recombinantpolynucleotide into the plant genome.

In general it is useful to introduce recombinant DNA randomly, i.e. at anon-specific location, in the genome of a target plant line. In specialcases it may be useful to target recombinant DNA insertion in order toachieve site-specific integration, for example to replace an existinggene in the genome, to use an existing promoter in the plant genome, orto insert a recombinant polynucleotide at a predetermined site known tobe active for gene expression. Several site specific recombinationsystems exist which are known to function implants include cre-lox asdisclosed in U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S.Pat. No. 5,527,695.

Transformation methods of this invention are preferably practiced intissue culture on media and in a controlled environment. “Media” refersto the numerous nutrient mixtures that are used to grow cells in vitro,that is, outside of the intact living organism. Recipient cell targetsinclude, but are not limited to, meristem cells, callus, immatureembryos and gametic cells such as microspores, pollen, sperm and eggcells. It is contemplated that any cell from which a fertile plant maybe regenerated is useful as a recipient cell. Callus may be initiatedfrom tissue sources including, but not limited to, immature embryos,seedling apical meristems, microspores and the like. Cells capable ofproliferating as callus are also recipient cells for genetictransformation. Practical transformation methods and materials formaking transgenic plants of this invention, for example various mediaand recipient target cells, transformation of immature embryo cells andsubsequent regeneration of fertile transgenic plants are disclosed inU.S. Pat. Nos. 6,194,636 and 6,232,526, which are incorporated herein byreference.

The seeds of transgenic plants can be harvested from fertile transgenicplants and be used to grow progeny generations of transformed plants ofthis invention including hybrid plants line for selection of plantshaving an enhanced trait. In addition to direct transformation of aplant with a recombinant DNA, transgenic plants can be prepared bycrossing a first plant having a recombinant DNA with a second plantlacking the DNA. For example, recombinant DNA can be introduced intofirst plant line that is amenable to transformation to produce atransgenic plant which can be crossed with a second plant line tointrogress the recombinant DNA into the second plant line. A transgenicplant with recombinant DNA providing an enhanced trait, e.g. enhancedyield, can be crossed with transgenic plant line having otherrecombinant DNA that confers another trait, for example herbicideresistance or pest resistance, to produce progeny plants havingrecombinant DNA that confers both traits. Typically, in such breedingfor combining traits the transgenic plant donating the additional traitis a male line and the transgenic plant carrying the base traits is thefemale line. The progeny of this cross will segregate such that some ofthe plants will carry the DNA for both parental traits and some willcarry DNA for one parental trait; such plants can be identified bymarkers associated with parental recombinant DNA, e.g. markeridentification by analysis for recombinant DNA or, in the case where aselectable marker is linked to the recombinant, by application of theselecting agent such as a herbicide for use with a herbicide tolerancemarker, or by selection for the enhanced trait. Progeny plants carryingDNA for both parental traits can be crossed back into the female parentline multiple times, for example usually 6 to 8 generations, to producea progeny plant with substantially the same genotype as one originaltransgenic parental line but for the recombinant DNA of the othertransgenic parental line.

In the practice of transformation DNA is typically introduced into onlya small percentage of target plant cells in any one transformationexperiment. Marker genes are used to provide an efficient system foridentification of those cells that are stably transformed by receivingand integrating a transgenic DNA construct into their genomes. Preferredmarker genes provide selective markers which confer resistance to aselective agent, such as an antibiotic or herbicide. Any of theherbicides to which plants of this invention may be resistant are usefulagents for selective markers. Potentially transformed cells are exposedto the selective agent. In the population of surviving cells will bethose cells where, generally, the resistance-conferring gene isintegrated and expressed at sufficient levels to permit cell survival.Cells may be tested further to confirm stable integration of theexogenous DNA. Commonly used selective marker genes include thoseconferring resistance to antibiotics such as kanamycin and paromomycin(nptII), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) orresistance to herbicides such as glufosinate (bar or pat) and glyphosate(aroA or EPSPS). Examples of such selectable are illustrated in U.S.Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and 6,118,047. Selectablemarkers which provide an ability to visually identify transformants canalso be employed, for example, a gene expressing a colored orfluorescent protein such as a luciferase or green fluorescent protein(GFP) or a gene expressing a beta-glucuronidase or uidA gene (GUS) forwhich various chromogenic substrates are known.

Plant cells that survive exposure to the selective agent, or plant cellsthat have been scored positive in a screening assay, may be cultured inregeneration media and allowed to mature into plants. Developingplantlets regenerated from transformed plant cells can be transferred toplant growth mix, and hardened off, for example, in an environmentallycontrolled chamber at about 85% relative humidity, 600 ppm CO₂, and25-250 microeinsteins m⁻² s⁻¹ of light, prior to transfer to agreenhouse or growth chamber for maturation. Plants are regenerated fromabout 6 weeks to 10 months after a transformant is identified, dependingon the initial tissue. Plants may be pollinated using conventional plantbreeding methods known to those of skill in the art and seed produced,for example self-pollination is commonly used with transgenic corn. Theregenerated transformed plant or its progeny seed or plants can betested for expression of the recombinant DNA and selected for thepresence of enhanced agronomic trait.

Transgenic Plants and Seeds

Transgenic plants derived from the plant cells of this invention aregrown to generate transgenic plants having an enhanced trait as comparedto a control plant and produce transgenic seed and haploid pollen ofthis invention. Such plants with enhanced traits are identified byselection of transformed plants or progeny seed for the enhanced trait.For efficiency a selection method is designed to evaluate multipletransgenic plants (events) including the recombinant DNA, for examplemultiple plants from 2 to 20 or more transgenic events. Transgenicplants grown from transgenic seed provided herein demonstrate improvedagronomic traits that contribute to increased yield or enhanced waterdeficit tolerance or both.

Not all transgenic events will be in transgenic plant cells that provideplants and seeds with an enhanced or desired trait depending on factors,such as location and integrity of the recombinant DNA, copy number,unintended insertion of other DNA, etc. As a result transgenic plantcells of this invention are identified by screening transformed progenyplants for enhanced water deficit stress tolerance and yield. Forefficiency a screening program is designed to evaluate multipletransgenic plants preferably with a single copy of the recombinant DNAfrom 2 or more transgenic events.

The following examples are included to demonstrate embodiments of theinvention. It should be appreciated by those of skill in the art thatthe techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention, therefore all matter set forth or shown in the accompanyingdrawings and examples is to be interpreted as illustrative and not in alimiting sense.

EXAMPLE 1

This example describes construction of plant expression vectors used fortransforming plant cells useful in the various aspects of the invention.Plasmid maps of such DNA constructs are illustrated in FIGS. 1 and 2,where the plasmid of FIG. 1 is used for transforming monocot plants suchas corn and the plasmid of FIG. 2 is used for transforming dicot plantssuch as soybean. Each plasmid contains a NF-YB expression cassette and aglyphosate herbicide resistance expression cassette within Agrobacteriumtumefaciens T-DNA borders identified as LB and RB respectively. Eachplasmid also contains origins of replication and repressor elements forreplication in cells (oriV, rop and oriColE) and aspectinomycin/streptomycin bactericidal selectable marker (SPC/STR).

With particular reference to FIG. 1 plasmid pMON82754 contains betweenleft and right Agrobacterium T-DNA borders (LB and RB) recombinant DNAconstructs comprising an NF-YB protein expression cassette and aglyphosate resistance expression cassette. The NF-YB expression cassettehas a promoter element (SEQ ID NO:8) which comprises a corn bundlesheath enhanced promoter and a transcription enhancing intron from acorn heat shock protein 70 gene followed by the DNA encoding a cornNF-YB protein (SEQ ID NO:44) and a 3′ element from Agrobacteriumtumefaciens transcript 7. The glyphosate resistance expression cassettecomprises a rice actin 1 promoter, leader and intron operably linked toa DNA encoding a chloroplast transit peptide from an Arabidopsisthaliana EPSPS gene and DNA encoding an EPSPS from an A. tumefaciensgene (CP4) and a 3′ element from an A. tumefaciens nopaline synthasegene. Other plasmids are prepared in which the promoter element and DNAencoding the NF-YB element are replaced with each of the promoterelements identified in Table 1 and each of the DNA encoding NF-YBprotein elements identified in Table 2. Thus, separate plasmids fortransforming monocot plant cells have recombinant DNA constructs forexpressing an NF-YB protein where each promoter identified in Table 1 isoperably linked to each of the DNAs encoding an NF-YB protein identifiedin Table 2. Monocot plant cells from corn, switchgrass, rice, andsugarcane are transformed with each of the plasmids producing multipletransgenic events of each recombinant DNA construct; and transgenicplants are regenerated and grown to produce transgenic seed or propagule(in the case of sugarcane) for each of the transgenic events.

With reference to FIG. 2, plasmid pMON63796 contains a recombinant DNAconstruct for expressing NF-YB protein with an enhanced CaMV35S promoter(SEQ ID NO:1) operably linked to DNA encoding the native Arabidopsisthaliana NF-YB protein identified as G481 (SEQ ID NO:53). The plasmid isused to produce transgenic dicot cells in which the Arabidopsis NF-YBprotein is ubiquitously expressed by the promoter. Other plasmids areprepared in which the promoter element is replaced with each of thepromoter elements identified in Table 1 and the DNA encoding the NF-YBelement is replaced with each of the DNA encoding NF-YB protein elementsidentified in Table 2. Thus, separate plasmids for transforming dicotplant cells have recombinant DNA constructs for expressing an NF-YBprotein where each promoter identified in Table 1 is operably linked toeach DNA encoding NF-YB protein identified in Table 2. Canola, alfalfa,cotton and soybean plant cells are transformed with each of the plasmidsproducing multiple transgenic events of each recombinant DNA construct;and transgenic plants are regenerated and grown to produce transgenicseed for each of the transgenic events.

Many transgenic events which survive to fertile transgenic plants thatproduce seeds and progeny plants will not exhibit the traits of waterdeficit stress tolerance and enhanced yield. Screening of progenytransgenic plants is necessary to identify a transgenic plant cell ofthis invention. Transgenic plants having enhanced water deficittolerance are identified from populations of plants transformed asdescribed herein by evaluating the trait in a variety of water deficitassays. More specifically, after transformation transgenic plants arepropagated to produce seed or propagules and homozygous progeny plantsare identified and screened for water deficit tolerance (e.g. usingmethods described below) to identify plants that produce seed of thisinvention.

The plants of this invention are screened for water deficit tolerance ascompared to control plants (tested as inbreds or hybrids) by ahigh-throughput method of greenhouse screening following waterwithholding, called “drought treatment”. For example, the greenhousescreen for transgenic corn plants for water use efficiency measureschanges in plant growth rate, e.g., at least a 10% improvement, inheight and biomass during a vegetative drought treatment, as compared tocontrol plants. The hydration status of the shoot tissues following thedrought is also measured. Shoot Initial Height (SIH) is plant heightafter 3 weeks of growth under optimum conditions. Shoot Wilt Height(SWH) is plant height at the end of a 6 day drought. Time courseexperiments have shown that at about 3 days of drought treatment, wildtype corn plants basically stop growing and begin to wilt. Thus atransgenic corn plant with improved water use efficiency will continueto grow (possibly to a lesser extent than with water) and thereby end upas a significantly taller plant at the end of a drought experiment.Shoot Wilt Mass (SWM) is the amount of wet and dry matter in the shoot(plant separated from root ball at the soil line) at the end of thedrought; SDM is measure after 2 to 3 weeks in a drying chamber. ShootTurgid mass (STM) is the SWM plus the mass of the water that istransported into plant tissues in 3 days of soaking in 40 degree Celsiuswater in the dark. Experiments have shown that most of the water ispulled up in 24 hours but it takes 2 more days before additionalincrease becomes insignificant. STM-SWM is indicative of water useefficiency in plants where recovery from stress is more important thanstress tolerance per se. Relative water content (RWC) is a measurementof how much (%) of the plant is water at harvest.RWC=(SWM−SDM)/(STM−SDM)*100. Fully watered corn plants are about 98%RWC. Typically, in a wilt screen the plants are about 60% RWC. Plantswith higher RWC at the end of a drought are considered to be healthierplants and more fit for post-drought recovery and growth. RelativeGrowth Rate (RGR) is calculated for each shoot using the formulaRGR=(SWH−SIH)/((SWH+SIH)/2)*100. Similar screening following a droughttreatment is done for transgenic canola, cotton and soybean plants.

Events of transgenic corn plants expressing a modified NF-YB proteinfrom DNA of SEQ ID NO:29-43 show improved water deficit stress toleranceas compared to wild type control plants and transgenic control plantsexpressing a natural NF-YB protein.

Events of transgenic corn plants expressing a native NF-YB protein fromDNA of SEQ ID NO: 44 or 45 operably linked to a promoter selected fromSEQ ID NO: 1-27 show improved water deficit stress tolerance as comparedto wild type control plants.

EXAMPLE 2

This example describes yield analysis results for transgenic plantsdisclosed in US 2005/0022266 A1. The disclosed plants comprise arecombinant transcription unit comprising a low level-expressing riceactin promoter operably linked to DNA coding for an NF-YB (SEQ ID NO:28)protein followed by a transcription unit comprising a CaMV 35S promoteroperably linked to DNA coding for the nptII marker (pMON73605).

Table 3A demonstrates that these plants exhibit enhanced yield (bu/acre)under water deficit stress, as evidenced by two consecutive years oftesting. TABLE 3A Improved Yield Under Water Deficit Stress pMON73605 -rice actin 1 promoter: NF-YB2 (expression enhanced by cis elements) Year1 Water Water Year 2 Deficit Deficit Water Deficit Stress Stress StressWater Deficit Event Yield Delta p-value Yield Delta Stress p-valueZM_M23837 13.7 .012 16.6 0.13 ZM_M25520 31.2 0.00 15.9 0.13 ZM_M2420754.8 <.001 16.9 0.11

Table 3B presents yield results (bu/acre) under sufficient waterconditions and demonstrates that the yield of pMON73605 transgenicplants under sufficient water conditions is significantly decreased.TABLE 3B Reduced Yield Under Sufficient Water Conditions Year 1 Year 2Sufficient Sufficient Sufficient Water Sufficient Water Water EventYield Delta Water p-value Yield Delta p-value ZM_M23837 −54.9 <.001 −23<.001 ZM_M25520 −40.3 <.001 −28 <.001 ZM_M24207 −51.3 <.001 −35 <.001

NFY B protein levels determined for these transgenic events are providedin Table 3C below. TABLE 3C NF-YB2 protein levels V12 to VT leaf proteinpg NF-YB2/microgram total Event protein ZM_M23837 61.4 ZM_M25520 51.8ZM_M24207 61.0

The CaMV 35S promoter in this construct is able to enhance the otherwiselow expression of the nearby rice actin promoter resulting in theproduction of greater than 40 picograms of NF-YB2 protein per microgramof total protein in the plant leaf tissue. The high level of protein inthe transgenic plants results in a reduction in yield when the plantsare grown under water sufficient conditions. In contrast, enhancerlessrice actin/NFYB expression constructs (pMON82452 and pMON82453) aredescribed below which produce less than 20 pg NF-YB2/microgram totalleaf protein and transgenic plants having enhanced yield under bothwater deficit stress conditions and sufficient water conditions aredescribed.

EXAMPLE 3

Transgenic corn plants prepared as described in Example 1 comprising DNAconstructs stably inserted in the chromosome and expressing an NF-YBprotein under the control of promoters shown in Table 4 were evaluatedfor yield under water deficit stress and sufficient conditions. TABLE 4Promoter::NF-YB Constructs in Transgenic Corn Plants Promoter PromoterSEQ ID Protein Vector Enhanced CaMV 35S SEQ ID NO: 2 NFB2 PMON84654Rubisco activase SEQ ID NO: 8 NFB2 PMON82754 Rice tubulin SEQ ID NO: 13NFB2a pMON82753 Rice tubulin SEQ ID NO: 13 NFB2 pMON82752 rab17 SEQ IDNO: 9 NFB2 PMON82454 Enhancerless rice actin SEQ ID NO: 15 NFB2aPMON82453 Enhancerless rice actin SEQ ID NO: 14 NFB2 PMON82452 p326 SEQID NO: 18 NFB2 PMON78305 FDA/PPDK SEQ ID NO: 27 NFB2 PMON78304 PPDK SEQID NO: 10 NFB2 PMON78303 CaMV 35S SEQ ID NO: 3 NFB2 PMON73611 NAS SEQ IDNO: 16 NFB2 PMON73610

These transgenic corn plants were analyzed for yield under water-deficitstress conditions, for yield under water sufficiency conditions and foramounts of NF-YB protein expressed in leaf tissue.

Homozygous inbred corn plants with recombinant DNA as described inExample 1 were crossed with compatible tester lines to produce hybridseed. The resulting seed was advanced to replicated yield trials ingeographical regions where corn is conventionally grown, e.g. in thestates of Iowa, Illinois, Kansas and California. In some trials, fieldwater content was controlled by irrigation, while other trials relied onnatural rainfall. Transgenic events advanced to this study werepre-selected to be single copy for the selectable marker associated withthe transgene. Control and transgenic events were planted at the sameplant density and replication. Field management of plant pest, weeds,tillage and fertilization was consistent with geography specificpractices.

In irrigated fields, the transgenic and control plants were irrigated tobe within field water-holding capacity until V10 corn leaf stage.Irrigation water was delivered via drip irrigation or overhead linearirrigation. To provide water deficit stress conditions, once the cornplants reached the V10 leaf stage, water was allowed to be limitinguntil plants demonstrated significant AM leaf rolling for 2 consecutivedays. The duration of this water regime spanned the V10 leaf through theR2 reproductive stage. Once the crop reached the R2 developmental stage,watering was resumed to full recovery through the remaining growingseason.

Once the corn crop reached physiological maturity, i.e. 10-25% grainmoisture, plots were harvested. Resulting grain yield was normalized to15.5% moisture and expressed in terms of bushels/acre.

Yield data analysis was performed for water deficit stress andsufficient water conditions. The yields were analyzed in several ways.One approach involved the use of statistical cluster analysis to selectgroups of locations having similar environmental characteristicspertaining to drought. The three variables used to form clusters ofsimilar locations were: the average daily high temperature during thethirty days before and thirty days after flowering; the averagedifference between cumulative precipitation and applied water versusevapotranspiration during the same sixty-day period, weighted by theproximity to flowering using the standard normal distribution to defineweights; and the average yield of a control pedigree at the location.Another analysis of variance model was then used to analyze yields, withfixed effects for clusters and random effects for the locations nestedwithin their corresponding clusters.

The yield data analysis compared the yields of all events from a singleconstruct as compared to corresponding control plots. Events were alsocompared to a corresponding control for event level analysis. Results ofthe yield analysis (Yield Deltas shown as bu/ac) are reported in Tables5A to 5M. TABLE 5A pMON82753 - rice alpha tubulin promoter SEQ ID NO 13:NF-YB2a Water Sufficient Water Deficit Deficit Water Sufficient StressStress Event Yield Delta Water p-value Yield Delta p-value ZM_M860674.5106 0.266 16.0421 0.011 ZM_M83476 −6.6879 0.116 11.763 0.08 ZM_M84797−3.6472 0.38 11.7199 0.064 ZM_M83475 2.8742 0.478 5.4529 0.364 ZM_M847382.1377 0.624 1.7943 0.789 ZM_M83470 −2.3375 0.862 1.088 0.871 ZM_M84105−15.0532 <.001 0.4693 0.944 ZM_M84086 1.4321 0.736 −2.1618 0.78ZM_M83465 0.3697 0.927 −4.4721 0.456 ZM_M86087 −11.0371 0.006 −5.84710.33 ZM_M84741 −11.3175 0.006 −7.9079 0.211 ZM_M83478 −5.2598 0.195−8.0171 0.182 ZM_M86065 −8.7812 0.355 −8.8923 0.215

TABLE 5B pMON82454 - rab17 promoter SEQ ID NO 9: NF-YB2 Water SufficientWater Deficit Deficit Water Sufficient Stress Stress Event Yield DeltaWater p-value Yield Delta p-value ZM_S112714 0.3879 0.924 −12.0102 0.036ZM_S112682 0.7015 0.863 −5.8292 0.357 ZM_S110587 −3.2144 0.428 −4.63130.441 ZM_S112667 −7.003 0.084 −3.7511 0.512 ZM_S111896 −3.5696 0.401−1.992 0.728 ZM_S112713 10.2417 0.445 0.09028 0.989 ZM_S110594 −4.31890.287 0.4137 0.945 ZM_S112696 −6.3222 0.128 0.4887 0.935 ZM_S112654−8.3365 0.045 0.867 0.88 ZM_S112701 −5.4576 0.178 0.9761 0.865ZM_S112657 −0.4083 0.976 1.0792 0.865

TABLE 5C pMON82754 - corn bundle sheath promoter SEQ ID NO 8: NF-YB2Water Sufficient Water Deficit Deficit Water Sufficient Stress StressEvent Yield Delta Water p-value Yield Delta p-value ZM_S110144 −6.98260.085 13.5813 0.024 ZM_S110890 −4.9583 0.522 10.6975 0.111 ZM_S110843−2.2576 0.578 4.5413 0.449 ZM_S110837 −3.3767 0.427 3.8793 0.54ZM_S110106 −12.4485 0.002 1.0213 0.865 ZM_S110893 −7.8212 0.054 0.29130.961 ZM_S111476 −3.6553 0.367 −0.2487 0.967 ZM_S110134 −10.1762 0.014−3.3787 0.574 ZM_S110873 −0.419 0.92 −6.0041 0.343 ZM_S110119 −6.15980.129 −14.7387 0.014

TABLE 5D pMON84654 - enhancedCaMV35S promoter SEQ ID NO2: NF-YB2 WaterSufficient Water Deficit Deficit Water Sufficient Stress Stress EventYield Delta Water p-value Yield Delta p-value ZM_S119096 −46.7705 <.001−0.09792 0.988 ZM_S117108 −43.9468 <.001 −11.8074 0.062 ZM_S119061−37.014 <.001 −11.8317 0.049 ZM_S119030 −44.4451 <.001 −12.8967 0.032ZM_S119088 −48.1538 <.001 −13.1315 0.038 ZM_S119034 −48.0195 <.001−13.5567 0.024 ZM_S119047 −52.5443 <.001 −13.8889 0.028 ZM_S117101−41.4371 <.001 −14.4537 0.022 ZM_S117341 −55.5489 <.001 −15.3574 0.015ZM_S119099 −43.7741 <.001 −15.7417 0.009 ZM_S119033 −45.9945 <.001−18.9417 0.002 ZM_S119077 −31.7358 <.001 −20.6117 <.001 ZM_S117346−15.6325 0.099

TABLE 5E pMON82752 - rice alpha tubulin promoter SEQ ID NO13: NF-YB2Water Sufficient Sufficient Deficit Water Water Stress Water DeficitEvent Yield Delta p-value Yield Delta Stress p-value ZM_M83933 −8.830.033 14.363 0.017 ZM_M82773 0.4403 0.916 10.4845 0.067 ZM_M84408−0.8068 0.853 7.0527 0.218 ZM_M82770 −14.2708 0.193 5.5578 0.438ZM_M82769 −2.0203 0.763 5.2828 0.461 ZM_M84389 −1.4877 0.714 3.92090.494 ZM_M82855 −35.2312 0.009 3.4116 0.59 ZM_M83306 −1.217 0.832 0.96140.893 ZM_M84393 0.8677 0.834 −5.182 0.388 ZM_M83321 3.2487 0.423 −5.72460.317 ZM_M82772 −0.8763 0.829 −5.952 0.322 ZM_M85712 −10.5414 0.022−6.5328 0.302

TABLE 5F pMON82453 - rice actin1 promoter SEQ ID NO15: NF-YB2a WaterSufficient Sufficient Deficit Water Water Stress Water Deficit EventYield Delta p-value Yield Delta Stress p-value ZM_M87052 −3.9922 0.401−7.594 0.258 ZM_M88601 6.2671 0.131 −4.0602 0.499 ZM_M87033 1.4629 0.718−0.9602 0.873 ZM_M88602 5.8765 0.147 −0.8908 0.894 ZM_M88129 0.37650.926 0.01482 0.998 ZM_M87027 2.2129 0.585 0.3398 0.955 ZM_M87051−4.2189 0.298 0.6598 0.912 ZM_M87036 −1.8401 0.657 4.072 0.52 ZM_M873780.797 0.844 5.772 0.362 ZM_M88595 6.2552 0.132 6.5848 0.273 ZM_M870494.1652 0.304 7.6698 0.201 ZM_M87335 0.4311 0.915 9.5915 0.13

TABLE 5G pMON82452 - rice actin1 promoter SEQ ID NO 14: NF-YB2 WaterSufficient Sufficient Deficit Water Water Stress Water Deficit EventYield Delta p-value Yield Delta Stress p-value ZM_M87949 5.1553 0.2156.5758 0.299 ZM_M85725 −9.5685 0.021 1.1316 0.851 ZM_M87438 −9.35180.021 0.7814 0.902 ZM_M87019 0.5323 0.896 −0.7982 0.9 ZM_M87010 0.60170.885 −1.5234 0.8 ZM_M87952 −2.5268 0.533 −2.5019 0.693 ZM_M85731−1.7604 0.759 −3.2605 0.627 ZM_M87441 1.9918 0.64 −4.7293 0.481ZM_M85734 −3.2154 0.428 −4.8284 0.421 ZM_M87937 0.03685 0.993 −6.89340.251 ZM_M87427 1.0596 0.794 −7.5684 0.208 ZM_M87000 −0.4518 0.911−8.2884 0.168 ZM_M87936 −6.7261 0.202 −8.3605 0.213

TABLE 5H pMON78305 - P326 promoter SEQ ID 18: NF-YB2 Water SufficientSufficient Deficit Water Water Stress Water Deficit Event Yield Deltap-value Yield Delta Stress p-value ZM_S121122 0.8762 0.833 4.1907 0.508ZM_S121068 −0.7 0.863 4.0241 0.525 ZM_S121130 −0.05682 0.989 3.79630.549 ZM_S121124 2.6568 0.512 2.0417 0.734 ZM_S121091 5 0.218 −3.55930.574 ZM_S121092 −0.1214 0.977 −5.4083 0.368 ZM_S121070 −2.819 0.497−5.75 0.364 ZM_S121096 2.6024 0.531 −7.7981 0.218 ZM_S121064 3.52050.385 −11.0033 0.067 ZM_S121123 4.4341 0.274 −11.7033 0.051

TABLE 5I pMON78304 - FDA/PPDK promoter SEQ ID NO 27: NF-YB2 WaterSufficient Sufficient Deficit Water Water Stress Water Deficit EventYield Delta p-value Yield Delta Stress p-value ZM_S120150 −15.8444 <.001−0.53 0.93 ZM_S120028 −14.4106 <.001 −0.9697 0.866 ZM_S119399 −8.57620.039 −6.0833 0.288 ZM_S119395 −9.2742 0.022 −7.9389 0.21 ZM_S120146−15.7765 <.001 −9.3407 0.14 ZM_S120046 −16.665 <.001 −12.3067 0.04

TABLE 5J pMON78303 - PPDK promoter SEQ ID 10: NF-YB2 Water SufficientSufficient Deficit Water Water Stress Water Deficit Event Yield Deltap-value Yield Delta Stress p-value ZM_S114670 −5.0583 0.212 9.3727 0.139ZM_S114672 −8.8856 0.028 5.9254 0.324 ZM_S116983 −18.1152 <.001 5.84040.331 ZM_S115592 −12.9152 0.001 5.4604 0.363 ZM_S115605 5.2875 0.693ZM_S115597 −10.8538 0.007 5.1095 0.372 ZM_S115611 −11.1083 0.006 4.13040.492 ZM_S115590 −14.397 <.001 −0.8846 0.883 ZM_S117062 −13.6379 <.001−5.3746 0.371 ZM_S114676 −9.1311 0.024 −6.0133 0.294 ZM_S114678 −14.2968<.001 −6.8546 0.254 ZM_S117016 −7.45 0.496 −14.662 0.029

TABLE 5K pMON73611 - CaMV35S promoter SEQ ID NO 3: NF-YB2 WaterSufficient Sufficient Deficit Water Water Stress Water Deficit EventYield Delta p-value Yield Delta Stress p-value ZM_S115348 −12.6042 0.25−0.1982 0.978 ZM_S114565 −23.2198 <.001 −0.6462 0.923 ZM_S114577−16.5312 <.001 −2.6359 0.645 ZM_S114556 −17.9972 <.001 −4.072 0.498ZM_S115373 −20.5903 <.001 −4.7313 0.409 ZM_S115260 −26.2934 <.001−4.7775 0.476 ZM_S114535 −19.7153 <.001 −6.122 0.308 ZM_S114591 −25.0631<.001 −8.4177 0.142 ZM_S115266 −14.0919 <.001 −12.0466 0.057 ZM_S115340−17.9381 <.001 −15.3295 0.011 ZM_S115350 −21.8187 0.104 −15.4681 0.021ZM_S115261 −21.3938 0.111 −20.2605 0.001

TABLE 5L pMON73610 - corn root promoter SEQ ID NO 16: NF-YB2 WaterSufficient Sufficient Deficit Water Water Stress Water Deficit EventYield Delta p-value Yield Delta Stress p-value ZM_S115519 3.9933 0.3498.0867 0.297 ZM_S115721 1.7023 0.675 7.19 0.283 ZM_S114691 6.5114 0.1085.4689 0.388 ZM_S115769 8.8727 0.029 5.242 0.383 ZM_S114480 4.2227 0.2994.172 0.487 ZM_S115567 −2.3 0.716 4.091 0.569 ZM_S117076 1.7811 0.7172.6267 0.714 ZM_S115703 −2.05 0.613 −0.6922 0.913

TABLE 5M pMON73605 - rice actin 1 promoter: NF-YB2 (expression enhancedby cis elements) Water Sufficient Sufficient Deficit Water Water WaterStress Deficit Stress Event Yield Delta p-value Yield Delta p-valueZM_M23837 −23.1583 <.001 −20.7881 <.001 ZM_M24207 −21.8297 <.001−12.3592 0.057 ZM_M25520 −25.9283 <.001 −15.8775 0.023 ZM_M26961−23.7417 <.001 −10.1582 0.106 ZM_M26962 −24.8995 <.001 −12.6188 0.052

To correlate expression of NF-YB protein with water-deficit toleranceand yield under water deficit and optimal water conditions, NF-YB2protein was measured in the transgenic plants by standard ELISAtechniques using polyclonal antibodies raised in rabbit. NF-YB2 proteinlevel is reported as “picograms of NF-YB2 protein per microgram of totalprotein” and includes both native and exogenous NF-YB2 protein. Totalprotein was measured using the Bradford protein assay (Bio-Rad,Hercules, Calif.). Background levels of NF-YB2 protein (pg NF-YB2/μgtotal protein) in each of the tissues measured are as follows: V3 leaf3.5 V12 leaf 6 Root 2.5 Silk 5 Tassel 3.2 Kernel 9.1 Immature cob 22.6

Results of analysis of protein levels in various tissues and at variousstages of development are reported in Tables 6A and 6B as the average ofmultiple events for each construct. TABLE 6A pg NF-YB2/μg total proteinPromoter Yield Leaf Leaf Leaf Leaf aver- Construct sequence table (V3)(V12) (V15) (VT-R1) age PMON84654 2 5D 107.0 121.9 195.3 136.4 144.4PMON78303 10 5J 71.6 83.1 141.5 112.1 106.9 PMON73611 3 5K 66.0 67.8127.1 79.6 88.8 PMON78304 27 5I 49.1 56.2 108.7 73.4 75.1 PMON73605 155M 39.5 57.7 79.6 54.2 62.2 PMON82754 8 5C 36.5 35.4 64.5 32.6 43.3PMON78305 18 5H 7.8 5.1 5.3 4.7 5.2 PMON82752 13 5E 6.6 4.9 6.5 5.5 5.6PMON82452 14 5G 6.0 7.8 10.5 8.7 8.8 PMON73610 16 5L 5.2 3.9 4.5 3.5 4.0PMON82454 9 5B 1.1 9.2 11.4 5.5 8.5

TABLE 6B pg NF-YB2/μg total protein Im- Promoter yield Ker- matureConstruct sequence table Root Silk Tassel nel Cob PMON84654 2 5D 4.6 9.633.6 33.2 149.6 PMON78303 10 5J 4.9 7.0 12.3 18.4 26.8 PMON73611 3 5K1.8 18.4 42.9 32.4 59.4 PMON78304 27 5I 3.3 2.9 50.7 21.4 28.3 PMON7360515 5M 15.6 38.0 20.8 22.2 60.7 PMON82754 8 5C 3.2 6.9 11.5 19.3 44.4PMON78305 18 5H 1.7 5.4 10.3 15.8 40.1 PMON82752 13 5E 4.5 4.3 9.8 28.0106.2 PMON82452 14 5G 3.1 3.5 66.8 31.6 38.8 PMON73610 16 5L 3.5 21.67.9 33.5 36.7 PMON82454 9 5B 3.9 2.8 4.7 19.6 32.0

Results of the analysis of data for yield under water-deficit stressconditions, yield under water sufficiency conditions and amounts ofNF-YB protein expressed in leaf tissue (corrected to substractbackground level of NF-YB protein produced from the native DNA) forindividual events is presented in Table 7 (as compared to the aggregatedconstruct level data presented in Table 6A and 6B). The data demonstratean inverse correlation between the level of NF-YB protein expressed andenhanced yield under both water-deficit stress conditions and sufficientwater conditions. When yield (bu/acre) is plotted against NF-YB levels,events that show enhanced yield under water-deficit stress conditions,also contained low levels of NF-YB (up to 40 picograms of NF-YB2 proteinper microgram of total protein in the plant leaf tissue) (FIG. 3).Similarly, events that show enhanced yield (Yield Deltas shown asbu/acre) under water sufficient conditions also contained low levels ofNF-YB (up to 40 picograms of NF-YB2 protein per microgram of totalprotein in the plant leaf tissue) (FIG. 4). An especially usefulembodiment for enhanced yield in corn under a wide range of availablewater conditions provides 0.1 to 11 pg NFB2/ug total protein. Particularexamples are ZM_M87949 (8.0 pg NFB2/ug total protein; +6.6 bu/acre yieldincrease under water-deficit stress; +5.2 bu/acre yield increase underwater sufficiency) and ZM_M88595 (7.5 pg NFB2/ug total protein; +6.6bu/acre yield increase under water-deficit stress; +6.3 bu/acre yieldincrease under water sufficiency). TABLE 7 Yield and Protein Data fromCorn Containing Promoter::NF-YB Constructs Water Sufficient Deficit pgNF-YB2/ Water Stress μg total Yield Yield Promoter Vector Event proteinDelta Delta Enhanced PMON84654 ZM_S117101 181.5 −41.4 −14.5 CaMV 35SEnhanced PMON84654 ZM_S117341 177.3 −55.5 −15.4 CaMV 35S EnhancedPMON84654 ZM_S119096 170.7 −46.8 −0.1 CaMV 35S Enhanced PMON84654ZM_S119034 168.1 −48.0 −13.6 CaMV 35S Enhanced PMON84654 ZM_S119033165.6 −46.0 −18.9 CaMV 35S Enhanced PMON84654 ZM_S119061 164.8 −37.0−11.8 CaMV 35S Enhanced PMON84654 ZM_S117108 158.7 −43.9 −11.8 CaMV 35SEnhanced PMON84654 ZM_S119088 157.2 −48.2 −13.1 CaMV 35S EnhancedPMON84654 ZM_S119099 143.7 −43.8 −15.7 CaMV 35S Enhanced PMON84654ZM_S119030 139.3 −44.4 −12.9 CaMV 35S Enhanced PMON84654 ZM_S119047136.0 −52.5 −13.9 CaMV 35S PPDK PMON78303 ZM_S115590 123.8 −14.4 −0.9PPDK PMON78303 ZM_S114678 121.0 −14.3 −6.9 CaMV 35S PMON73611 ZM_S114565117.1 −23.2 −0.6 PPDK PMON78303 ZM_S115592 110.8 −12.9 5.5 EnhancedPMON84654 ZM_S119077 110.4 −31.7 −20.6 CaMV 35S PPDK PMON78303ZM_S115597 107.4 −10.9 5.1 PPDK PMON78303 ZM_S115611 107.1 −11.1 4.1PPDK PMON78303 ZM_S114670 106.4 −5.1 9.4 PPDK PMON78303 ZM_S114672 105.4−8.9 5.9 CaMV 35S PMON73611 ZM_S114591 104.7 −25.1 −8.4 PPDK PMON78303ZM_S116983 104.6 −18.1 5.8 Enhanced PMON84654 ZM_S117346 104.3 −15.6 NotCaMV 35S determined PPDK PMON78303 ZM_S117062 102.3 −13.6 −5.4 CaMV 35SPMON73611 ZM_S115340 102.1 −17.9 −15.3 CaMV 35S PMON73611 ZM_S115260100.9 −26.3 −4.8 PPDK PMON78303 ZM_S115605 100.8 Not 5.3 determined CaMV35S PMON73611 ZM_S115373 97.2 −20.6 −4.7 CaMV 35S PMON73611 ZM_S11453592.8 −19.7 −6.1 Rubisco PMON82754 ZM_S110893 86.9 −7.8 0.3 activase CaMV35S PMON73611 ZM_S114577 86.8 −16.5 −2.6 PPDK PMON78303 ZM_S114676 83.0−9.1 −6.0 FDA/PPDK PMON78304 ZM_S120046 76.7 −16.7 −12.3 FDA/PPDKPMON78304 ZM_S120146 75.8 −15.8 −9.3 FDA/PPDK PMON78304 ZM_S120028 75.1−14.4 −1.0 CaMV 35S PMON73611 ZM_S115261 74.7 −21.4 −20.3 PPDK PMON78303ZM_S117016 74.4 −7.5 −14.7 FDA/PPDK PMON78304 ZM_S119395 72.2 −9.3 −7.9CaMV 35S PMON73611 ZM_S115266 72.1 −14.1 −12.0 CaMV 35S PMON73611ZM_S115350 71.7 −21.8 −15.5 FDA/PPDK PMON78304 ZM_S120150 70.8 −15.8−0.5 CaMV 35S PMON73611 ZM_S115348 69.6 −12.6 −0.2 FDA/PPDK PMON78304ZM_S119399 64.9 −8.6 −6.1 CaMV 35S PMON73611 ZM_S114556 61.3 −18.0 −4.1Rubisco PMON82754 ZM_S110843 53.8 −2.3 4.5 activase Rubisco PMON82754ZM_S110119 48.7 −6.2 −14.7 activase Rubisco PMON82754 ZM_S110134 46.6−10.2 −3.4 activase Rubisco PMON82754 ZM_S110106 44.5 −12.4 1.0 activaseRubisco PMON82754 ZM_S110144 43.4 −7.0 13.6 activase Rubisco PMON82754ZM_S110890 42.0 −5.0 10.7 activase Rubisco PMON82754 ZM_S110837 37.0−3.4 3.9 activase Rubisco PMON82754 ZM_S111476 37.0 −3.7 −0.2 activaseEnhancerless PMON82453 ZM_M87051 16.1 −4.2 0.7 rice actin rab17PMON82454 ZM_S112701 15.7 −5.5 1.0 Enhancerless PMON82452 ZM_M87438 14.8−9.4 0.8 rice actin Enhancerless PMON82452 ZM_M87010 14.3 0.6 −1.5 riceactin rab17 PMON82454 ZM_S112696 11.1 −6.3 0.5 rab17 PMON82454ZM_S111896 10.9 −3.6 −2.0 Enhancerless PMON82453 ZM_M88601 10.8 6.3 −4.1rice actin Enhancerless PMON82452 ZM_M87952 10.5 −2.5 −2.5 rice actinrab17 PMON82454 ZM_S112667 10.3 −7.0 −3.8 rab17 PMON82454 ZM_S11058710.2 −3.2 −4.6 rab17 PMON82454 ZM_S110594 10.1 −4.3 0.4 EnhancerlessPMON82453 ZM_M87033 9.9 1.5 −1.0 rice actin rab17 PMON82454 ZM_S1126829.7 0.7 −5.8 Enhancerless PMON82453 ZM_M88129 9.5 0.4 0.0 rice actinEnhancerless PMON82452 ZM_M87937 9.5 0.0 −6.9 rice actin rab17 PMON82454ZM_S112657 9.2 −0.4 1.1 Enhancerless PMON82452 ZM_M87441 9.2 2.0 −4.7rice actin rab17 PMON82454 ZM_S112654 9.1 −8.3 0.9 Rice tubulinpMON82752 ZM_M82769 9.0 −2.0 5.3 Enhancerless PMON82452 ZM_M87000 8.9−0.5 −8.3 rice actin Enhancerless PMON82452 ZM_M87019 8.8 0.5 −0.8 riceactin Enhancerless PMON82452 ZM_M85731 8.8 −1.8 −3.3 rice actinEnhancerless PMON82453 ZM_M87036 8.7 −1.8 4.1 rice actin EnhancerlessPMON82453 ZM_M87027 8.4 2.2 0.3 rice actin Enhancerless PMON82452ZM_M85725 8.4 −9.6 1.1 rice actin rab17 PMON82454 ZM_S112713 8.3 10.20.1 Enhancerless PMON82453 ZM_M87049 8.1 4.2 7.7 rice actin EnhancerlessPMON82453 ZM_M87378 8.0 0.8 5.8 rice actin Enhancerless PMON82452ZM_M87949 8.0 5.2 6.6 rice actin Enhancerless PMON82452 ZM_M85734 8.0−3.2 −4.8 rice actin Rice tubulin pMON82753 ZM_M86065 7.8 −8.8 −8.9 Ricetubulin pMON82752 ZM_M84408 7.6 −0.8 7.1 Enhancerless PMON82453ZM_M88602 7.6 5.9 −0.9 rice actin Rice tubulin pMON82752 ZM_M82855 7.5−35.2 3.4 Rice tubulin pMON82752 ZM_M85712 7.5 −10.5 −6.5 EnhancerlessPMON82453 ZM_M88595 7.5 6.3 6.6 rice actin Rice tubulin pMON82753ZM_M83475 7.4 2.9 5.5 Enhancerless PMON82453 ZM_M87335 7.4 0.4 9.6 riceactin Rice tubulin pMON82753 ZM_M84105 7.2 −15.1 0.5 Rice tubulinpMON82753 ZM_M84738 7.0 2.1 1.8 Rice tubulin pMON82753 ZM_M84086 7.0 1.4−2.2 rab17 PMON82454 ZM_S112714 7.0 0.4 −12.0 Enhancerless PMON82452ZM_M87427 7.0 1.1 −7.6 rice actin Rice tubulin pMON82753 ZM_M84741 6.9−11.3 −7.9 Enhancerless PMON82453 ZM_M87052 6.9 −4.0 −7.6 rice actinEnhancerless PMON82452 ZM_M87936 6.8 −6.7 −8.4 rice actin Rice tubulinpMON82753 ZM_M86087 6.7 −11.0 −5.8 Rice tubulin pMON82752 ZM_M83321 6.53.2 −5.7 Rice tubulin pMON82753 ZM_M83478 6.4 −5.3 −8.0 Rice tubulinpMON82752 ZM_M82773 6.4 0.4 10.5 Rice tubulin pMON82752 ZM_M83306 6.4−1.2 1.0 Rice tubulin pMON82752 ZM_M84389 6.2 −1.5 3.9 Rice tubulinpMON82752 ZM_M84393 6.1 0.9 −5.2 p326 PMON78305 ZM_S121096 6.1 2.6 −7.8Rice tubulin pMON82753 ZM_M86067 5.9 4.5 16.0 Rice tubulin pMON82753ZM_M83476 5.9 −6.7 11.8 Rubisco PMON82754 ZM_S110873 5.8 −0.4 −6.0activase Rice tubulin pMON82753 ZM_M83470 5.8 −2.3 1.1 p326 PMON78305ZM_S121092 5.8 −0.1 −5.4 NAS PMON73610 ZM_S115519 5.8 4.0 8.1 Ricetubulin pMON82752 ZM_M83933 5.7 −8.8 14.4 p326 PMON78305 ZM_S121064 5.73.5 −11.0 Rice tubulin pMON82753 ZM_M84797 5.6 −3.6 11.7 Rice tubulinpMON82752 ZM_M82772 5.5 −0.9 −6.0 p326 PMON78305 ZM_S121124 5.5 2.7 2.0p326 PMON78305 ZM_S121122 5.4 0.9 4.2 p326 PMON78305 ZM_S121123 5.4 4.4−11.7 p326 PMON78305 ZM_S121068 5.3 −0.7 4.0 NAS PMON73610 ZM_S1146915.3 6.5 5.5 Rice tubulin pMON82753 ZM_M83465 5.1 0.4 −4.5 p326 PMON78305ZM_S121130 5.0 −0.1 3.8 Rice tubulin pMON82752 ZM_M82770 4.7 −14.3 5.6NAS PMON73610 ZM_S115769 4.6 8.9 5.2 NAS PMON73610 ZM_S114480 4.4 4.24.2 NAS PMON73610 ZM_S117076 4.4 1.8 2.6 NAS PMON73610 ZM_S115703 4.4−2.1 −0.7 NAS PMON73610 ZM_S115567 4.0 −2.3 4.1 p326 PMON78305ZM_S121070 3.9 −2.8 −5.8 NAS PMON73610 ZM_S115721 3.9 1.7 7.2 p326PMON78305 ZM_S121091 3.6 5.0 −3.6

EXAMPLE 4

Transgenic cotton plants prepared as described in Example 1 comprisingDNA constructs stably inserted in the chromosome and expressing an NF-YBprotein under the control of promoters shown in Table 8 are evaluatedfor yield under water deficit stress and sufficient conditions. TABLE 8Promoter::NF-YB Constructs in Transgenic Cotton Plants Promoter PromoterSEQ ID Protein Vector Enhanced CaMV 35S SEQ ID NO: 2 ArabidopsispMON83103 G481 rd29a SEQ ID NO: 19 Arabidopsis pMON95538 G481 Tsfl SEQID NO: 60 Arabidopsis pMON95559 G481

Events of transgenic cotton plants comprising the above constructs andexpressing the Arabidopsis NF-YB protein are grown under water deficitstress and sufficient water conditions and events are identified thathave low leaf protein levels which impart improved yield (lbs/acre) ascompared to wild type control plants when grown under water deficitstress conditions and comparable or improved yield as compared to wildtype control plants when grown under sufficient water conditions.

EXAMPLE 5

Transgenic soybean plants prepared as described in Example 1 comprisingDNA constructs stably inserted in the chromosome and expressing an NF-YBprotein under the control of promoters shown in Table 9 are evaluatedfor yield under water deficit stress and sufficient conditions. TABLE 9Promoter::NF-YB Constructs in Transgenic Soybean Plants PromoterPromoter SEQ ID Protein Vector Soybean phaseolin SEQ ID NO: 61Arabidopsis pMON106646 G481 Enhanced CaMV 35S SEQ ID NO: 2 SoybeanpMON83057 G481-6 Enhanced CaMV 35S SEQ ID NO: 2 Arabidopsis pMON63796G481

Events of transgenic soybean plants comprising the above constructs andexpressing the Arabidopsis or soybean G481 NF-YB proteins are grownunder water deficit stress and sufficient water conditions and eventsare identified that have low leaf protein levels which impart improvedyield yield (bu/acre) as compared to wild type control plants when grownunder water deficit stress conditions and comparable or improved yieldas compared to wild type control plants when grown under sufficientwater conditions.

EXAMPLE 6

Transgenic alfalfa, canola, switchgrass, sugarcane and rice plantsprepared as described in Example 1 comprising DNA constructs stablyinserted in the chromosome and expressing an NF-YB protein under thecontrol of promoters shown in Table 1 are evaluated for yield underwater deficit stress and sufficient water conditions. Events of thesetransgenic plants are grown under water deficit stress and sufficientwater conditions and events are identified that have low leaf proteinlevels which impart improved yields compared to wild type control plantswhen grown under water deficit stress conditions and comparable orimproved yield as compared to wild type control plants when grown undersufficient water conditions.

All of the materials and methods disclosed and claimed herein can bemade and used without undue experimentation as instructed by the abovedisclosure. Although the materials and methods of this invention havebeen described in terms of preferred embodiments and illustrativeexamples, it will be apparent to those of skill in the art thatvariations may be applied to the materials and methods described hereinwithout departing from the concept, spirit and scope of the invention.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

1. A plant chromosomal DNA segment comprising a recombinantpolynucleotide flanked by native plant DNA, wherein said polynucleotideprovides for expression of at least an NF-YB protein and a markerprotein, and wherein said NF-YB protein is produced in leaf cells ofsaid plant at a level up to 40 picograms per microgram of total proteinin said plant leaf tissue cells.
 2. A plant chromosomal DNA segmentcomprising a recombinant polynucleotide flanked by native plant DNA,wherein said polynucleotide provides for expression of a single protein,wherein said protein is an NF-YB protein, and wherein said NF-YB proteinis produced in leaf cells of said plant at a level up to 40 picogramsper microgram of total protein in said plant leaf tissue cells.
 3. Aplant chromosomal DNA segment comprising a recombinant DNA construct forexpressing an NF-YB protein comprising contiguous amino acids from SEQID NO:28, wherein said amino acids include: (a) amino acids at position49 thorough 122 of SEQ ID NO:28 and wherein one or more amino acids atposition number 49, 73, 76, 83, 89, 102, 103, 109, 115, 118 or 122 aredifferent; or (b) amino acids at position 49 thorough 122 of SEQ ID NO:28 and wherein one or more amino acids at position number 49, 73, 76,83, 89, 102, 103, 109, 115, 118 or 122 are different and one or more ofamino acids at position 55 through 61 are missing; (c) amino acids ofSEQ ID NO: 28 at position 29 through 134 and one or more of amino acidsat position 2 through 28 are missing; or (d) amino acids of SEQ ID NO:28 at position 68 through 134 and one or more of amino acids at position2 through 67 are missing.
 4. A plant chromosomal DNA segment of any ofclaims 1-3 wherein said recombinant polynucleotide comprises a promoteris selected from the group consisting of a rice alpha tubulin promoter,a rice actin promoter, a PPDK mesophyl tissue enhanced promoter, and arubisco activase bundle sheath enhanced promoter.
 5. A plant chromosomalDNA segment of any of claims 1-3 and wherein said NF-YB protein isproduced in leaf cells of said plant at a level between 0.1 and 11picograms per microgram of total protein in said plant leaf tissuecells.
 6. A transgenic plant cell comprising a plant chromosomal DNAsegment of any of claims 1-3.
 7. A transgenic crop of water deficitstress tolerant plants comprising cells of claim 6 wherein the harvestedyield of said crop is comparable to or enhanced over the yield of a cropof control plants not having said plant chromosomal DNA segment whensaid crops are grown in water sufficient conditions.
 8. A transgeniccrop of claim 7 wherein said water deficit stress tolerant plants arecorn, cotton, soybean, sugarcane, switchgrass, rice, wheat, alfalfa, orcanola plants.
 9. A transgenic corn plant seed comprising a plantchromosomal DNA segment of any of claims 1-3 wherein said NF-YB proteinis a native corn protein.
 10. A method of improving water stresstolerance and yield in a crop plant line comprising providing in thegenome of said crop plant line a plant chromosomal DNA segment of any ofclaims 1-3.
 11. A transgenic pollen grain comprising a haploidderivative of a plant cell containing a a plant chromosomal DNA segmentof any of claims 1-3.
 12. A method for manufacturing non-natural,transgenic seed that can be used to produce a crop of transgenic plantswith enhanced water deficit stress tolerance resulting from expressionof an NF-YB protein from a plant chromosomal DNA segment of any ofclaims 1-3, wherein said method comprises: (a) screening a population ofplants having said plant chromosomal DNA segment and control plants forsaid enhanced yield when grown under deficit stress or enhanced orcomparable yield as compared to the yield for control plants when grownunder sufficient water conditions, (b) selecting from said populationone or more plants that exhibit enhanced yield as compared to the yieldfor control plants under water deficit stressed or enhanced orcomparable yield as compared to the yield for control plants when grownunder water sufficient conditions, (c) verifying that said plantchromosomal DNA segment is stably integrated in said selected plants,(d) analyzing leaf tissue of a selected plant to determine theproduction of transgenic NF-YB protein at a level up to 40 picograms ofNF-YB protein per microgram of total protein in said leaf tissue; and(e) collecting seed or a regenerative propagule from a selected plant.13. A method of claim 12 wherein said seed is corn, cotton, soybean,sugarcane, switchgrass, rice, wheat, alfalfa, or canola seed.
 14. Amethod of producing inbred corn seed comprising: (a) acquiring hybridcorn seed from a herbicide tolerant corn plant which also hasstably-integrated, chromosomal DNA segment of any of claims 1-3; (b)introgressing the chromosomal DNA segment from said acquired hybrid cornseed into a second corn line by allowing pollen grains comprising ahaploid derivative with said chromosomal DNA segment to pollinate saidsecond corn line to produce crossed seeds, (c) producing a population ofplants from crossed seeds wherein a fraction of the seeds produced fromsaid pollination is homozygous for said chromosomal DNA segment, afraction of the plants produced from said hybrid corn seed is hemizygousfor said chromosomal DNA segment, and a fraction of the plants producedfrom said hybrid corn seed does not have said chromosomal DNA segment;(d) selecting corn plants which are homozygous and hemizygous for saidchromosomal DNA segment by treating with an herbicide; (e) collectingseed from herbicide-treated-surviving corn plants and planting said seedto produce further progeny corn plants; (f) backcrossing plants grownfrom said progeny seeds with said second corn line to produce an inbredcorn line.
 15. The method of claim 14 further comprising crossing saidinbred corn line with a third corn line to produce hybrid seed. 16.Anti-counterfeit milled seed having, as an indication of origin, a plantcell with said chromosomal DNA segment of any of claims 1-3.
 17. Amethod of growing a corn, cotton, soybean, sugarcane, switchgrass, rice,wheat, alfalfa, or canola crop without irrigation water comprisingplanting seed having plant cells with a plant chromosomal DNA segment ofany of claims 1-3 which are selected for enhanced water deficittolerance.